This invention relates generally to the family of hepatitis GB viruses (HGBV) and more particularly, relates to reagents such as antisense nucleic acid sequences and methods utilizing these nucleic acid sequences which are useful for controlling translation of HGBV-A, -B, or -C, both in vivo and in vitro, by either increasing or decreasing the expressions of HGBV-A, -B or -C proteins.
Recently, a new family of flaviviruses detected in patients with clinically diagnosed hepatitis was reported. This new family of viruses has been named the "GB" viruses, after the initials of the patient first infected with the virus. These viruses have been reported by J. N. Simons et al., Proc. Natl. Acad. Sci. USA 92:3401-3405 (1995); and J. N. Simons et al., Nature Medicine 1(6):564-569 (1995). Three members of the family have been identified to date: GBV-A, GBV-B and GBV-C. T. P. Leary, et al., J. Med. Virol. 48:60-67 (1995) While HGBV-A appears at this time to be of non-human primate source, HGBV-C is clearly of human source. Currently, the source of HGBV-B is unknown. These viruses are thought to play a role in transmittable hepatitis disease of viral origin.
The GB viruses appear to be members of the Flaviviridae family. They possess RNA genomes approximately 9.5 kb in size which contain a single long open reading frame (ORF). Structural and nonstructural proteins are encoded in the N-terminal one-third and C-terminal two-thirds of the putative viral polyproteins, respectively. Phylogenetic analyses of the nonstructural helicase and replicase genes demonstrate that these viruses are related to, but distinct from, the HCV genus of the Flaviviridae. See, for example, T. P. Leary, et al., supra and A. S. Muerhoff et al., J. Virol. 69:5621-5630 (1995). Specifically, GBV-A and GBV-C appear most closely related as they share a common ancestor, while the GBV-A/C ancestor, GBV-B and HCV all appear to be equally divergent from other members of the Flaviviridae.
However, when the 5' nontranslated regions (NTRs) and structural genes are examined, a more striking division between the GB viruses and the other members of the Flaviviridae becomes apparent. GBV-B appears similar to the HCV and pestivirus genera of the Flaviviridae. Conserved sequences present in the 5' NTRs of HCV and pestiviruses are found in the 5' NTR of GBV-B, and GBV-B and HCV share closely related RNA secondary structures within the 5' NTR. (M. Honda, E. A. Brown and S. M. Lemon, manuscript submitted).
Moreover, a basic (pI=11.1) core protein is present at the N-terminus of the GBV-B putative polyprotein precursor, and two putative envelope glycoproteins with several potential N-linked glycosylation sites arc located downstream of core in GBV-B. A. S. Muerhoff et al., supra. These structural proteins appear in all members of the Flaviviridae examined to date. See, for example, M. S. Collett et al., J. Gen. Virol. 69:2637-2643 (1989) and R. H. Miller and R. H. Purcell, Proc. Natl. Acad. Sci. USA 87:2057-2061 (1990).
In contrast to GBV-B, examination of GBV-A and GBV-C reveals marked differences between these viruses and other genera of the Flaviviridae. GBV-A and GBV-C contain long 5' NTRs that have limited sequence identity at the 5' NTR to each other but no identity to the 5' NTRs GBV-B, HCV or pestiviruses. GBV-A and GBV-C also encode putative envelope proteins that contain relatively few potential N-linked glycosylation sites. Most strikingly, clearly discernible basic core proteins are not found in the cDNA sequences cloned thus far from these viruses.
The absence of core proteins would distinguish GBV-A and GBV-C from other genera of the Flaviviridae. However, several important aspects of the structure of the GBV-A and GBV-C genomes remain undefined. Primary among these is the identification of the AUG codons at which translation of the viral polyproteins initiate. The sequence of GBV-A contains two potential in-frame initiator AUG codons 27 nucleotides (9 amino acids) and immediately upstream of the putative E1 signal sequence. Similarly, multiple GBV-C sequences possess two to three potential in-frame initiator AUG codons. See, T. P. Leary et al., supra: and J. Linnen et al., Science 271:505-508 (1996). However, none of these AUGs have been demonstrated to serve as the initiator codon, and initiation at any of these sites would result in a severely truncated core protein at best. It is conceivable that deletions during the cloning of these virus RNAs could have resulted in the elimination of core sequences or a disruption of the true ORF in this region of the genome, as suggested by Leary et al., supra. However, multiple RT-PCR products generated from the 5' ends of GBV-A and GBV-C using a variety of primers, polymerases and conditions (unpublished data), in addition to determining the 5' end sequences of over 35 separate GBV-C isolates (U.S. Ser. No. 08/580,038, filed Dec. 21, 1995, previously incorporated herein by reference) provide no support for the existence of additional sequence missing from the previously described cDNA clones. Thus, it is possible that the 5' ends of these viruses are complete (or nearly complete), and that GBV-A and GBV-C do not encode core proteins.
Of the genera that comprise the Flaviviridae, the viruses classified in the flaviviruses genus (e.g., yellow fever virus, dengue virus) contain relatively short 5' NTRs of 97 to 119 nucleotides. In these viruses, translational initiation is thought to utilize a conventional eukaryotic ribosome scanning mechanism in which ribosomes bind the RNA at a 5' cap structure and scan in a 3' direction until encountering an AUG codon in a favorable context for initiation. See, M. Kozak, Cell 44:283-292 (1989) and M. Kozak, J. Cell. Biol. 108:229-241 (1989).
In contrast to the flavivirus genus, genomic RNAs from members of the pestiviruses and HCV genera contain relatively long 5' NTRs of 341 to 385 nucleotides which in some ways are similar to those of picornaviruses. Extensive studies of the picornavirus 5' NTRs reveal that translation initiation occurs through a mechanism of internal ribosome entry. R. J. Jackson et al., Mol. Biol. Reports 19:147-159 (1994); K. Meerovitch and N. Sonnenberg, Semin. Virol. 67:3798-3807 (1993). This internal entry requires a defined segment of the viral 5' NTR known as an "internal ribosome entry site" (IRES) or "ribosome landing pad." The RNA comprising the cis-acting IRES forms highly ordered structures which interact with trans-acting cellular translation factors to bind the 40S ribosome subunit at an internal site on the viral message, often many hundreds of nucleotides downstream of the 5' end of the molecule. Such translation initiation functions in a 5' cap-independent fashion, and is generally not influenced by structure or sequence upstream of the IRES.
Practically, the ability of a sequence to function as an IRES is assessed by insertion of the sequence between two cistrons of a bicistronic RNA. If the intercistronic sequence contains an IRES, there is significant translation of the downstream cistron which is generally independent of the translational activity of the upstream cistron. Studies of the 5' NTRs of HCV and pestiviruses using bicistronic mRNAs demonstrate the presence of IRESs in these sequences. See, for example, T. L. Poole et al., Virology 206:750-754 (1995); R. Rijnbrand et al., FEBS Letters 365:115-119 (1995); K. Tsukiyama-Kohara et al., J. Virol. 66:1476-1483 (1992); and C. Wang et al., J. Virol. 67:3338-3344 (1993).
Structural changes in the IRES influence the rate of translation initiation. Thus, by modifying a virus' s IRES, one can control the amount of viral protein being made. Control of the translation process of the nucleic acids of GB viruses could provide an effective means of treating viral disease. The ability to control translation could result in a decrease of the expression of viral proteins. Also, the ability to increase expression may prove useful by producing greater amounts of GB viral proteins which could be utilized in a variety of ways, both diagnostically and therapeutically. Further, the ability to increase translation of the GB viruses in vivo may provide a means for increasing immune stimulation in an individual.
It therefore would be advantageous to provide reagents and methods for controlling the translation of HGBV proteins from HGBV nucleic acids. Such reagents would comprise antisense nucleic acid sequences or other compound which may specifically destabilize (or stabilize) the IRES structure. Such nucleic acid sequences or compounds could greatly enhance the ability of the medical community to provide a means for treating an individual infected with GB virus(es). In addition, IRESs are among the most highly consereved nuclcotide sequences. Identification of such a sequence immediately suggests a target for probe-based detection reagents. Diagnostic or screening tests developed from these reagents could provide a safer blood and organ supply by helping to eliminate GBV in these blood and organ donations, and could provide a better understanding of the prevalence of HGBV in the population, epidemiology of the disease caused by HGBV and the prognosis of infected individuals. Additionally, these consereved structures may provide a means for purifying GBV proteins for use in diagnostic assays.